Botanical Studies (2012) 53: 243-254. MORPHOLOGY

Stipules and colleters of the : morphology, structure and comparative significance

Chiou-Rong SHEUE1,*, Ying-Ju CHEN1, and Yuen-Po YANG2,3

1Department of Life Sciences, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung 402, Taiwan 2Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan 3Department of Bioresources, Dayeh University, 168 University Rd., Dacun, Changhua 515, Taiwan

(Received October 11, 2010; Accepted December 1, 2011)

ABSTRACT. One characteristic possessed by the entire mangrove Rhizophoraceae is the strong enclosure of its young shoots by conspicuous rounded or flattened stipules. Young are conspicuously immersed in resinous to milky exudates from colleters (multicellular external secretory emergences) located at the adaxial stipule bases. We systematically studied and compared morphological and structural features of stipules and their colleters from 18 taxa of the mangrove Rhizophoraceae. Three types of sclereid idioblasts ( and ), collenchymas, and thick cuticles, were found to provide a structural basis for the mechanical support of stipules. Several to hundreds (35-580) of finger-like colleters aggregate into -specific shapes: rectangular to trapezoidal (), triangular (Ceriops and ), or as a band (Rhizophora). Number of rows, total number of colleters per stipule, and their individual size, also vary by taxon, and have taxo- nomic value. All colleters of this subfamily are considered anatomically ‘standard’, regardless of whether they appear as short-stalked rods (Bruguiera), long-stalked rods (Ceriops, Kandelia) or acuminate rods (Rhizophora, no stalk). Colleters in these taxa are comprised of a central axis of slender, elongated cells and an outer pali- sade-like epidermis, with a secretory function, perpendicular to the axis. Based on stipule and colleter charac- teristics, Ceriops and Kandelia are closely related, but Rhizophora shows more derived features. The structural and mechanical protection provided by stipules and colleter exudates may help shield the young shoots of these mangrove from their harsh environments.

Keywords: Anatomy; Bruguiera; Ceriops; Colleter; Kandelia; ; Rhizophora; Sclereid; Standard type; Stipule.

INTRODUCTION the naked eye at the adaxial base of stipules in the of mangrove Rhizophoraceae (Tomlinson, 1986; Sheue Rhizophoraceae is well known as the richest mangrove et al., 2003a, b; Sheue et al., 2005). Except for the genus family, having four exclusively mangrove genera (Bru- Bruguiera, colleters, although few in number, are also guiera, Ceriops, Kandelia and Rhizophora) (Tomlinson, present within the bracteoles of mangrove Rhizophoraceae 1986). About 21 mangrove species of this family are inflorescences (Sheue, 2003; Sheue et al., 2003a). currently recognized, including new taxa from Kandelia Colleters are external secretory structures (Fahn, 1990), (Sheue et al., 2003b) and Ceriops (Sheue et al., 2009a, b; characterized by the production of a viscous substance Sheue et al., 2010). The family is diversified both ecologi- that covers, protects from desiccation, and lubricates the cally (habitat) and morphologically (trait). In addition to bud and young leaves of plants (Kronestedt-Robards and the mangroves mentioned above, the family has 15 inland Robards, 1991; Evert, 2007). Hou (1958) mentioned that genera with 135 species occurring in inland forests (Jun- colleters in Rhizophoraceae were first observed by Al- cosa and Tomlinson, 1988a). All members of the Rhizo- ston in African , and later by Metcalfe and phoraceae s. s. (excluding Anisophyllea) have large and Chalk in (both inland genera). These glandular conspicuous interpetiolar, glabrous and caducous stipules emergences inside the adaxial regions of stipules appear on both vegetative and reproductive shoots, strongly to occur in all Malaysian genera, both mangrove and in- ensheathing the young leaves and inflorescences (Hou, land (Hou, 1858). Colleter exudates from this family are 1958). Several to hundreds of aggregated finger-like col- assumed to be composed of phenolic compounds (Rosh- leters mixed with milky mucilage can be observed with china and Roshchina, 1993). Colleters have long been associated with a number of *Corresponding author: E-mail: [email protected]; Tel/ flowering plants, notably Apocynaceae and Rubiaceae. The Fax: +886-4-22857395. color and consistency of colleter exudates are variable in 244 Botanical Studies, Vol. 53, 2012 a somewhat diagnostic manner from species to species of MATERIALS AND METHODS the mangrove Rhizophoraceae (Tomlinson, 1986). How- ever, the knowledge of colleters of Rhizophoraceae is still materials containing stipules with colleters were under-reported and remains obscure. The presence of col- collected from 18 mangrove Rhizophoraceae taxa (includ- leters in R. mangle was noticed by Warming in 1883 and ing two hybrids of Rhizophora) from Asia and Australia briefly described (Gill and Tomlinson, 1969): “a feature of from 1999 to 2009 (Table 1). The voucher specimens were some physiological significance in the bud of Rhizophora deposited in the Herbarium of National Chung Hsing Uni- is a double row of yellow, glandular scales on the side of versity (TCB), Taichung, Taiwan. each stipule. The primodia enclosed by the stipules are Three individual plants were sampled from a popula- always bathed by a viscous fluid which seems to originate tion, and from each individual, three stipules were care- from these glands.” It is the only species in this family to fully detached from the shoot apices when the young have the colleters anatomically studied (Lersten and Cur- leaves were about to emerge from the stipules. Stipules tis, 1974). The main objectives of the present work are to were stored in 70% ethanol in the field, observed and pho- provide a systematic study of stipules and colleters of the tographed with a Zeiss dissecting microscope to determine mangrove species of this family in order to understand stipule morphology, number of colleters, and their forms their morphology, structure, taxonomic value, and com- of aggregation within the stipule. A portion of the materi- parative significance. als was observed with a S-2400 Scanning Electron Micro-

Table 1. Taxa and collection information of the mangrove species of Rhizophoraceae in this study. Species Collection locality Habitat (L.) Blume Pichavarum, Tamil Nadu, India Back or middle mangroves Sungei Buloh Wetland Reservoir, Singapore Ding Hou Cairns, Queensland, Australia Back or middle mangroves C. G. Rogers Pulau Ubin, Singapore Within mangroves (L.) Savigny Sungei Buloh Wetland Reservoir, Singapore Middle mangroves Cardwell, Queensland, Australia (Roxb.) W. & A. ex Pasir Ris Nature Park, Singapore Back or middle mangroves Griff. Cardwell, Queensland, Australia (Lour.) Poir. Benut, Johor, Malaysia Middle mangroves (C. T. White) Ballment, T. Cardwell, Queensland, Australia Back mangroves, landward J. Sm. & J. A. Stoddart Moreton Bay, Queensland, Australia (Griff.) Ding Hou Pichavarum, Tamil Nadu, India Back mangroves Ceriops pseudodecandra Sheue, H. Y. Liu, Darwin, Northern Territory, Australia Middle mangroves, along river bank C. C. Tsai and Yuen P. Yang Cairns, Queesland, Australia Back mangroves (Perr.) C. B. Rob. Sungei Buloh Wetland Reservoir, Singapore Within mangroves Cairns/ Cardwell, Queensland, Australia Back mangroves Ceriops zippeliana Blume Pasir Ris Nature Park, Singapore Back mangroves

Kandelia candel (L.) Druce Benut, Johor, Malaysia Malaysia Along river bank Sheue, H. Y. Liu & J. W. Tanshui, Taipei, Taiwan Along river bank, pure stand H. Yong Tongshi, Chaiyi, Taiwan River bank Blume Sungei Buloh Wetland Reservoir, Singapore Seaward mangroves Pichavarum, Tamil Nadu, India Cairns/ Cardwell, Queensland, Australia Rhizophora × annamalayana Kathiresan Pichavarum, Tamil Nadu, India Back mangroves Rhizophora × lamarckii Montr. Cairns/ cardwell, Queensland, Australia Back mangroves Lam. Pichavarum, Tamil Nadu, India Seaward mangroves Sungei Buloh Wetland Reservoir, Singapore Griff. Tainan, Taiwan Along salty canal Cairns/ Moreton Bay, Queensland, Australia Seaward mangroves SHEUE et al. — Stipules and colleters of the mangrove Rhizophoraceae 245 scope (Hitachi, Tokyo, Japan), after treating with ethanol series dehydration, critical point drying and gold coat- ing, allowing shape and size determination in particular. These stipules were also treated with the clearing method (Chiang, 1990) to investigate features such as sclereid id- iobalsts and crystals. For field fixation for anatomical study, the base of each stipule with colleters was cut into several small pieces and put into 1.25-1.5% glutaraldehyde in 0.1 M phosphate buf- fer with 5% sucrose. Materials were stored in this fixative for 5-7 days during collection abroad. For the second fixa- tion, materials were subsequently transferred to 1% OsO4 in 0.1 M phosphate buffer for about 4 hours. After dehy- dration through an ethanol series, materials were infiltrated Figure 1. The mangrove Rhizophoraceae with shoots and for 3 days and embedded within Spurr’s-resin. The embed- stipules. (A) The shoot apices and top nodes of Ceriops australis ded materials were polymerized in an oven at 70°C for with stipules (broad arrows), which are interpetiolar and cadu- 12 hours. Semi-thin sections (1 µm) were made by an Ul- cous. Primodia enclosed by the stipules are always bathed by a tracut E (Leica, Wetzlar, Germany) or a MTX Microtome viscous fluid. Young expanded leaves coated with resin-like mu- (RMC, Tucson, USA), stained with 0.1% Toluidine blue cilage (thin arrow); (B) A fresh stipule of Bruguiera exaristata for 1 minute or so, and then observed and photographed showing yellowish aggregated finger-like colleters with viscous with a BH-2 Light Microscope (Olympus, Tokyo, Japan). exudation at the adaxial base. Scale bar = 1 mm.

Table 2. Morphological and structural characters of stipules and colleters of the mangrove Rhizophoraceae, including aggregated form, number of rows and total number of colleters in each stipule, and individual shape and color of colleters. Stipule Colleter Taxon Morphology/ length (cm)/ Sc Aggregated form/ row no./ total no. Shape/ color Bruguiera cylindrica Round/ 3.0-4.0/ no Sc  / 4-7/ 70-90 SRs/ MW Bruguiera exaristata Round/ 2.0-3.0/ no Sc / 9-14/ 120-135 SRs/ MW-MY Bruguiera hainesii Round/ 3.5-4.2/ no Sc / 12-15/ 100-146 SRs/ MW Bruguiera gymnorhiza Round/ 4.0-5.0/ no Sc  / 9-13/ 200-220 SRs/ MW Bruguiera parviflora Round/ 4.5-6.5/ no Sc  / 6-8/ 160-170 SRs/ MW Bruguiera sexangula Round/ 4.5-5.5/ no Sc / 3-4 / 35-40 SRs/ MW Ceriops australis Flattened/ 1.0-1.2/ Sc 2 types ▲/ 9-11/ 90-140 SRl/ MW Ceriops decandra Flattened/ 1.2-2.4/ Sc 2 types ▲/ 7-8/ 50-70 SRl/ MW Ceriops pseudodecandra Flattened/ 2.0-3.0/ Sc 2 types ▲/ 8-12/ 80-100 SRl/ MW Ceriops tagal Flattened/ 1.5-2.8/ Sc 2 types ▲/ 24-26/ 165-205 SRl/ MW Ceriops zippeliana Flattened/ 2.5-3.6/ Sc 2 types ▲/ 18-20/ 280-310 SRl/ MW Flattened/ 3.0-4.0/ no Sc ▲/ 6-7/ 75-96 SRl/ MY Kandelia obovata Flattened/ 2.5-3.2/ no Sc ▲/ 8-9/ 95-110 SRl/ MY Rhizophora apiculata Round/ 5.0-8.5/ Sc 3 types ▬ / 2-4/ 160-200 ARse/ Y Rhizophora mucronata Round/ 4.5-6.5/ Sc 3 types ▬ / 6-9/ 460-580 ARse/ MY Rhizophora stylosa Round/ 4.5-5.5/ Sc 3 types ▬ / 4- 6/ 180-300 ARse/ MY Rhizophora × annamalayana Round/ 4.5-6.0/ Sc 3 types ▬ / 6-7/ 130-160 ARse/ MY Rhizophora × lamarckii Round/ 4.5-5.5/ Sc 3 types ▬ / 2-3/ 75-90 ARse/ MY Symbols: aggregated form: broad band ▬; narrow band ▬; rectangle ; trapezoid ; semicircle ; triangle ▲; Abbreviations: ARse-Sessile acuminate rod; MW-milky white; MY-milky yellow; Y-yellow; Sc: sclereid idioblast; SRl-long stalked rod; SRs- short stalked rod. 246 Botanical Studies, Vol. 53, 2012

Figure 2. Morphological features of stipules and colleters in mangrove Rhizophoraceae. Abbreviations: C: colleter, E: exudates, L: , S: stipule. (A) A shoot of Rhizophora apiculata with stipules, annular stipule scars and leaf scars; (B-C) Cross-section of a Ce- riops zippeliana shoot showing a pair of clasping stipules appearing as flattened type, with thickest part in the folding region and its thickness gradually thinning out toward its margin and apex; (C) A position closer to the base of the stipule with colleters. The con- volute young leaves, colleters and their exudates appear between the space of stipules; (D) Cross-section of a R. apiculata shoot with a pair of clasping stipules appearing as rounded type; (E-F) A Bruguiera gymnorhiza stipule with aggregated colleters forming a rect- angle at the adaxial base. Removing some stipule tissues reveals colleters in (F); (G) Aggregated colleters in B. cylindrica. Note that colleters are less compact at the distal end; (H) A broad band of aggregated colleters in R. mucronata; (I) Part of a colleter aggregation in R. apiculata. Scale bars: (A) and (D) = 5 mm; (B), (C), (E), (F), (G) and (H) = 1 mm; (I) = 500 μm. SHEUE et al. — Stipules and colleters of the mangrove Rhizophoraceae 247

RESULTS (Bruguiera and Rhizophora) (Figure 2A, 2D) and flattened (Ceriops and Kandelia, Figure 2B-C). The adaxial base of Stipule morphology each stipule contains a space filled with aggregated col- All the members of mangrove Rhizophoraceae have leters and a slightly sticky resin-like substance, which can distinct stipules paired outside young leaves (Figures 1A be observed from detached stipules (Figures 1B, 2E-I) or and 2A). Most of the stipules are green to yellow- green; stipule cross-sections (Figure 2B-C). Despite the morpho- however, some species (e.g. R. apiculata, R. stylosa, B. logical difference, clasping stipule vernation is present gymnorhiza) may have both red-purple and green stipules. in all species of the mangrove Rhizophoraceae, whereby The morphology of stipules is consistent within a genus, one margin is free and is thickest in the middle region and although size may vary interspecifically (Table 2). Two gradually thins out towards its edge (Figure 2B-D). types of stipule morphology can be recognized: rounded

Figure 3. Anatomical features of mangrove Rhizophoraceae stipules. Abbreviations: S: stipule, Sc: small sclereid idioblast, SSc: strands of aggregated sclereid idioblast. (A-B) Sclereid idioblasts in Rhizophora apiculata, several strands of aggregated sclereid idio- blasts (multibranched) and elongated rod idioblasts (arrow) parallel to the long axis of the stipules in (A); three isolated small sclereid idioblasts and aggregated sclereid idioblasts in (B); (C-D) Sclereid idioblasts in Ceriops australis, elongated rod idioblasts in (C) and an isolated small sclereid idioblast in (D); (E) Cross-section of R. stylosa stipule showing a strand of aggregated sclereid idioblasts; (F) The collenchyma in the base part of a stipule in Bruguiera parviflora, with a prismatic crystal inside a cell (arrow). Scale bars: (A), (B) and (E) = 100 μm; (C), (D) and (F) = 30 μm. 248 Botanical Studies, Vol. 53, 2012

Stipule structure idioblasts (Figure 3B) and elongated rod idioblasts, which The clearing method and cross-sections reveal that two are mostly parallel to the long axis of stipules (Figure 3A). genera of the mangrove Rhizophoraceae, Ceriops and In Ceriops, two kinds of sclereid idioblasts are observed: Rhizophora, have sclereid idioblasts inside the stipules. elongated rod idioblasts along the thickest mid region (Fig- Rhizophora has three types of sclereid idioblasts: strands of ure 3C) and isolated small idioblasts (Figure 3D). In con- aggregated sclereid idioblasts (Figure 3A-B), isolated small trast, Bruguiera and Kandelia have no sclereid idioblast in

Figure 4. SEM view of mangrove Rhizophoraceae colleters. Abbreviations: Cu: cuticle, PP: peripheral parenchyma, St: stalked rod colleter. (A- B) Bruguiera exaristata, colleters aggregate as a trapezoid form in (A) and an individual colleter appears as a short stalked rod in (B); (C) Ceriops decandra, with long stalked rod colleters; (D) Kandelia obovata, a part of the aggregated colleters in a trian- gular form; (E-F) Rhizophora apiculata; (E) Colleters appear as sessile acuminate rods; (F) A surface view of a colleter showing the detached cuticle and inner peripheral parenchyma cells. Scale bars: (A) = 1 mm; (B) and (C) = 100 μm; (D) and (E) = 500 μm; (F) = 20 μm. SHEUE et al. — Stipules and colleters of the mangrove Rhizophoraceae 249 their stipules, both instead possess collenchyma tissue near Figure 2H-I). Rhizophora apiculata colleters are bright the abaxial side of their stipules (Figure 3F). yellow, but those of other taxa of this genus are milky Stipule structure among mangrove Rhizophoraceae white. This feature could distinguish R. apiculata from species is similar, except for the presence of sclereid idio- other species of Rhizophora in the field. In addition, row blasts (Table 2, Figure 3E). Unlike in normal leaves, there numbers of colleters differ between species of this genus. is no differentiated palisade and spongy parenchyma in Rhizophora apiculata has just 2-3 rows of colleters, while these stipules. A thick cuticle covers the epidermis of both R. stylosa has 4-6, and R. mucronata has 6-9 (460-580 col- sides. The ground tissue consists mostly of collenchyma, leters). but parenchyma is found near the adaxial side. If pres- ent, sclereid idioblasts mostly occur within collenchyma. Individual shape and size of colleter Stipule tissues of mangrove Rhizophoraceae are also abun- Colleter attachment comes in both sessile and stalked dant in tannins (Figure 6A) and form druse (Figure 6A), or forms in the mangrove Rhizophoraceae (Table 2; Figures occasionally prismatic (Figure 3F), calcium oxalate crys- 4-5). Only the genus Rhizophora has sessile colleters (Fig- tals. ures 4E, 5D), while the colleters of Bruguiera, Ceriops and Kandelia have stalks near the base. Colleters of the Colleter species of Bruguiera appear as short stalked rods (stalk Several to numerous milky-white to yellowish colleters less than 30 µm long) with similar width from the base to of various shapes aggregated in multiple rows were found the tip (Figures 4B, 5A). Colleters of Ceriops and Kande- attached to the base of the adaxial side of the stipule (Fig- lia have long stalked rods, with stalks about or longer than ure 1B, Table 2). These colleters remain attached to the 100 µm in length (Table 3; Figures 4C, 5B-C). Colleters stipule, but shrink and gradually lose their fresh color as of Rhizophora are best described as sessile acuminate rods the stipule drops from the parent tree. However, colleters due to the uneven width (Figures 4E, 5D). may attach to the corresponding side of the stipule scar The size (most conveniently measured by length) of along with the basal residue of the stipule if the stipule is individual colleters may vary within the same aggregated removed while it is still strongly enclosing a shoot. Obvi- group within a stipule. For example, smaller colleters are ous secretions often glue the colleters together in all man- often observed near the base, but these smaller colleters grove taxa observed in this study. are relatively few in number. There are larger numbers of larger similarly-sized colleters within a stipule. These Aggregated form, rows and numbers of col- colleters are the basis of the comparative measurements leters in this study. It is clear that the genus Rhizophora has the A mass of colleters occurring at the adaxial base of largest colleters in these four genera of mangrove Rhizo- each stipule aggregates as a band, rectangle, trapezoid or phoraceae. The colleter size of six selected species of triangle. It is clear that these aggregated forms are generi- mangrove Rhizophoraceae is compared in Table 3 (stalks cally consistent (Table 2). included if stalks present). The colleters of R. stylosa reach 1008.9 ± 270 µm in length with a broad base (up to In Bruguiera, multirow colleters aggregate as a rect- angle or trapezoid (Figure 4A), and colleters located at the distal end of a stipule may appear more or less loosely organized (Figure 2G). There are from 3-14 rows, and c. 40-210 colleters in each stipule of the various Bruguiera species (Table 2). It is apparent that row number and total colleter number are species specific, and this could be a di- agnostic character for species differentiation. In the genus Bruguiera, B. gymnorhiza has the greatest number of col- leters (9-12 rows, 200-210 colleters) (Figure 2E-F), while B. sexangula has the least (3-4 rows, 35-40 colleters) (Table 2), although their stipules are nearly the same size. The stipules of Kandelia and Ceriops are flattened (Figure 2B-C) and both colleters are aggregated into a tri- angular shape (Figure 4D). Due to the tightly overlapping margins of the stipule, the colleters of these two genera are difficult to observe unless the stipule is cut into two halves. It is noteworthy that K. candel, with a larger stipule (3-4 cm long), has fewer colleters (75-96) than K. obovata, with a smaller stipule (2.5-3.2 cm long, 95-110 colleters) Figure 5. Diagram of the individual shapes of colleters in man- (Table 2). grove Rhizophoraceae stipules. (A) Bruguiera, short stalked rod; In the genus Rhizophora, colleters are aggregated either (B) Ceriops, long stalked rod; (C) Kandelia, long stalked rod; as narrow or broad bands, comprising 2-9 rows (Table 2; (D) Rhizophora, sessile acuminate rod. Scale bar = 100 µm. 250 Botanical Studies, Vol. 53, 2012

Figure 6. Anatomical structure of colleters in mangrove Rhizophoraceae stipules. Abbreviations: Cu: cuticle, CP: central core of parenchyma, PP: peripheral parenchyma, St: stalked rod colleter; T: Tanninferous cells. (A-B) Kandelia obovata; (C) Rhizophora stylosa. (A) A longitudinal section of a colleter showing the central core of elongated parenchyma, peripheral parenchyma and a stalk attached to a stipule. Abundant drused crystals (arrows) appear in the stipule tissue, but are absent from colleter cells. Tanninferous cells commonly occur in colleter and stipule core parenchyma, but not in the colleter epidermis; (B) Cross-section of a colleter showing central parenchyma surrounded by radically elongated peripheral epidermis; (C) A terminal part of a colleter showing an apparent space filled with exudates (arrow) between peripheral epidermis and the cuticle. Some exudates (starred) that drench young shoots are released after the rupture of the cuticle. All scale bars = 50 μm.

Table 3. Morphometric characters of colleters of six selected species of the mangrove Rhizophoraceae. Length1 (µm) Width (µm) Species N Stalk length (µm) (min) avg (max) ± std (min) avg (max) ± std

Bruguiera exaristata 12 (238) 406.6 (540) ± 106.6 (119) 153.3 (200) ± 27.7 Less than 30

Ceriops australis 10 (204) 371.2 (530) ± 100.8 (82) 97.9 (102) ± 9.1 160 ± 24

Ceriops decandra 10 (269) 516.0 (672) ± 119.9 (77) 157.2 (211) ± 46.1 92.7 ± 5.8

Ceriops tagal 10 (510) 674.8 (856) ± 115.2 (102) 130.6 (142) ± 18.2 Not measured

Kandelia obovata 10 (275) 500.5 (680) ± 129.2 (100) 155.8 (220) ± 39.2 119 ± 23.9 Rhizophora stylosa 10 (713) 1008.9 (1490) ± 270.0 Base2: 300.0 ± 28.2 No stalk Middle: 222.5 ± 11.6 Tip: 100.1 ± 18.1

1Stalks included if stalks present. 2Because colleter is an acuminate rod, widths were measured from the base, middle and tip. SHEUE et al. — Stipules and colleters of the mangrove Rhizophoraceae 251

300 µm) tapering to one-third of its basal width at the tip. generic level in the mangrove Rhizophoraceae. The num- Ceriops australis has the smallest colleters judged both by ber of colleter rows in an aggregation could have value at length (371.2 ± 100.8 µm) and by width (97.9 ± 9.1 µm). the specific level for most of these species. For example, In the genus Ceriops, colleter size varies in a gradation the five taxa of Ceriops appear very similar in morphol- (e.g. C. tagal: 674.8 ± 115.2 µm, C. decandra: 516.0 ± ogy (Sheue et al., 2009a, 2010). The characteristics of 119.9 µm, and C. australis: 371.2 ± 100.8 µm). colleters can serve as valuable vegetative diagnostic traits for Ceriops materials in the absence of reproductive parts. Structure of colleter This taxonomically valuable character has also been ap- Despite the variation in stalk presence and the size of plied to differentiate between species within Bruguiera colleters, the anatomical structure of colleters of all taxa (Sheue et al., 2005) and Kandelia (Sheue et al., 2003a, b). studied is remarkably similar. Viewed in transverse sec- A hand lens (c. 10X) is recommended to observe this fea- tion, colleters consist of a rather massive core of elongated ture in the field. parenchymatous cells (c. 8-10 rows) with unlignified walls The similarity in stipule and colleter morphology dem- in the center (Figure 6), sheathed by many peripheral pa- onstrates a close relationship between Ceriops and Kan- renchyma (palisade-like epidermal cells) arranged in one delia that is in agreement with the molecular phylogeny layer perpendicular to the axis (Figures 4F; 6A-B). Usually, (Setoguchi et al., 1999; Schwarzbach and Ricklefs, 2000). the cell walls of the core parenchyma are thicker than those However, two types of sclereid idioblasts are present in of the palisade-like epidermis. Many of these core paren- Ceriops stipules, but are absent from those of Kandelia. chyma cells are tanniferous. Drused crystals rarely occur No sclereid was found in Bruguiera or Kandelia stipules, in the parenchyma of the core region, and not at all in the but two and three sclereid types were found in Ceriops outer epidermal cells (Figure 6A). The outer thin-walled and Rhizophora stipules, respectively. This may imply that palisade-like epidermial cells are the secretory cells, with Ceriops and Rhizophora are more derived than the former dense cytoplasm. The mucilage accumulates in the inter- two genera (Bruguiera and Kandelia), with Rhizophora cellular spaces of epidermal cells as well as between the being the most derived genus in this subfamily. This result cuticle and the secretory epidermal cells (Figure 6C). is consistent with the phylogeny within this subfamily based on molecular (Setoguchi et al., 1999; Schwarzbach DISCUSSION and Ricklefs, 2000) and morphological evidence (Juncosa and Tomlinson, 1988b). All members of the mangrove Rhizophoraceae have In this study, colleters of two reported Rhizophora conspicuous stipules enclosing young shoots. However, hybrids were compared with colleters of their putative compared to leaf structure and function, stipules are much mother plants. As is usual, the morphological features of less studied and under-reported. In this study, we recog- hybrids were consistently intermediate to their putative nized three types of sclereid idioblasts in stipules of Ce- parents. Interestingly, only one hybrid showed an inter- riops and Rhizophora genera. The strands of aggregated mediate number of rows. Rhizophora × annamalayana sclereid idioblasts and the isolated small idioblasts in Kathiresan (Kathiresan, 1995), was reported from southern Rhizophora are astrosclereids, as reported for the stipules India and was established as a hybrid of R. apiculata and of R. mucronata (Shah and Sundarraj, 1965). This is the R. mucronata (Parani et al., 1997). The hybrid from India, first report of idioblasts in the Ceriops stipules. These iso- R.× annamalayana, has 5-6 rows of colleters, which ap- lated small idioblasts appear as short rods without branch pear as an intermediate between its parents (2-4 rows and arms. The elongated rod idioblasts are parallel to the long 6-9 rows ). In contrast, the hybrid, R. × lamarckii, reported axis of Ceriops stipules, similar to the arrangement of as a hybrid of R. apiculata (2-4 rows) and R. stylosa (4-6 elongated idioblasts along the leaf midrib in Rhizophora rows) (Duke and Bunt, 1979; Tomlinson, 1986), has 2-3 (Sheue, 2003). These idioblasts and collenchyma, along rows of colleters, which is closer to those of R. apiculata. with vascular tissues and the thick cuticle of stipules, pro- The ‘standard’ type colleter was first described by Ler- vide strong mechanical support that presumably protects sten (1974a), which is one of the widespread colleter types the shoot inside. in Rubiaceae. In addition, ‘dendroid’ and ‘brush-like’ types Both rounded and flattened stipules were recognized were described as Rubiaceous colleters (Lersten, 1974a, in the mangrove Rhizophoraceae. Stipule morphology b). The members of Apocynaceae also have standard type correlates with the shape of their contained colleter aggre- colleters intermingled with petiolar hairs on the adaxial gations. Flattened stipules appear in the two mangrove gen- side of the petiole (Thomas and Dave, 1991). In this study, era, Ceriops and Kandelia. These stipules are thickest in the colleters of all mangrove species of this family are the folding regions, and their thickness gradually thins out anatomically standard, as Lersten and Curtis (1974) ob- toward the margin and apex. As a consequence, a triangular served in R. mangle, and are remarkably similar to those space filled with colleters is formed in its adaxial base. found in several other families. However, various shapes The work reported here suggests that the forms of col- of colleters were recognized in this study: short-stalked leter aggregations, and the shapes of the individual col- rods (Bruguiera), long-stalked rods (Ceriops, Kandelia), leters in the stipules, could have taxonomic value at the or sessile acuminate rods (Rhizophora). The stalk structure 252 Botanical Studies, Vol. 53, 2012 of a colleter has seldom been reported, but Mandevilla is characterize their harsh environment. Since the colleters one example (Appezzato-da-Glória and Estelita, 2000). of this family are still under-reported, it would be inter- The foliar and intrapetiolar colleters of Mandevilla can esting to compare the differences between the inland and exhibit vascularization, but its interpetiolar colleters lack mangrove species with special reference to the ecological vascularization (Appezzato-da-Glória and Estelita, 2000). adaptations represented by stipule and colleter characters. Althought no vascularization similar to that observed for R. mangle (Lersten and Curtis, 1974) was found in the Acknowledgements. The authors thank G. T. Sundari, S. present study, we recently observed subtle vascularization Das and G. Agoramoothy for helping collect mangroves in K.obovata by more sensitive methods (Sheue et al., in in India, P. Saenger, G. Wightman, C. P. Mangion, and V. preparation). Sarafis for helping collect materials in Australia, J. Y. W. Lersten (1974a) found crystals in the colleters of the Yong, S. K. Lee and M. A. Rashid for helping collecting Rubiaceae. However, in the present study, an abundance materials in Singapore, and P. Chesson for improving the of drused crystals was observed within the stipule tissues manuscript. This study was supported by National Science beneath the colleters, but rarely inside colleters. This dif- Council (NSC-97-2126-B-005-002-MY3) of The Republic ference may imply differentiated cellular functions and of China (Taiwan). metabolic activities between colleters and stipules. Sig- nificant spaces between the peripheral parenchyma and LITERATURE CITED the cuticle containing a secretory product were commonly found in colleters of the mangrove Rhizophoraceae as ob- Appezzato-da-Glória, B. and M.E.M. Estelita. 2000. Develop- served in those of Gardenia (Mangalan et al., 1990). The ment, structure and distribution of colleters in Mandevilla secretory product is released after the rupture of the cuticle illustris and M. velutina (Apocynaceae). Rev. Bras. Bot. 23: and drenches the shoot apex inside the stipule (Mangalan 113-120. et al., 1990). Chiang, S.H.T. 1990. Principles of Plant Microtechnique. Maw- Rhizophora, a genus usually occurring at the front of Tsang, Taipei. mangroves facing the sea, has the largest colleters with Duke, N.C. and J.S. Bunt. 1979. The genus Rhizophora (Rhizo- an average length over 1 mm. The colleters of Ceriops, phoraceae) in north-eastern Australia. Aust. J. Bot. 27: 657- a back mangrove closer to land, are relatively smaller (c. 678. 370 μm in C. australis). The prominent size and numerous colleters of Rhizophora (460-580 in R. mucronata) repre- Evert, R.F. 2007. Esau’s Plant Anatomy: Meristems, Cells and sent the most developed colleters of this study. In addition, Tissues of the Plant Body: Their Structure, Function. 3rd the sessile acuminate rod of colleters in Rhizophora is edn. John Wiley & Sons. considered to be more derived in this family, since the col- Fahn, A. 1990. Plant Anatomy. 4th ed. Pergamon Press. leters of the other three genera are stalked. Gill, A.M. and P.B. Tomlinson. 1969. Studies on the growth of Erythroxylaceae was recently confirmed as a sister fam- red mangrove ( L.) I. Habit and general ily of Rhizophoraceae (Setoguchi et al., 1999; Schwarz- morphology. Biotropica 1: 1-9. bach and Ricklefs, 2000). Thiebaut and Hoffmann (2005) Hou, D. 1958. Rhizophoraceae. In C. G. G. J. van Steenis (ed.), described colleters in four genera of Erythroxylaceae, and Flora Malesiana, ser. 1, Vol. 5. Noordhoff- Kolff N. V., Dja- compared to two genera of Rhizophoraceae. Colleters of karta, pp. 429-473. Erythroxylaceae are of the same type as those of Rhizo- Juncosa, A.M. and P.B. Tomlinson. 1988a. A historical and taxo- phoraceae, but position, arrangement, shape and size differ nomic synopsis of Rhizophoraceae and Anisophylleaceae. between all examined genera (Thiebaut and Hoffmann, Ann. Missouri Bot. Gard. 75: 1278-1295. 2005). In addition, the authors commented that the obvi- ous secretion gluing the colleters together occurs in the Juncosa, A.M. and P.B. Tomlinson. 1988b. Systematic compari- mangrove R. mucronata, but not in the inland member of son and some biological characteristics of Rhizophoraceae the Rhizophoraceae, Cassipourea malosana (Thiebaut and and Anisophylleaceae. Ann. Missouri Bot. Gard. 75: 1296- Hoffmann, 2005). In terms of colleter number and size, it 1318. is evident that the mangrove Rhizophoraceae has more and Kathiresan, K. 1995. Rhizophora annamalayana: A new species larger colleters, as reported here, than those observed in of mangroves. Environ. Ecol. 13: 240-241. the inland Rhizophoraceae, Cassipourea malosana (Thie- Kronestedt-Robards, E. and A. Robards. 1991. Exocytosis in baut and Hoffmann, 2005), Carallia and gland cells. In C. Hawea, J. Coleman, and D. Evans (eds.), (Sheue, 2003). Endocytosis, Exocytosis and Vesicle Traffic in Plants. Soc. The combined evidence from morphological and ana- Exp. Biol. Seminar Series 45: 199-232. tomical features of these mangroves and observations in Lersten, N.R. and J.D. Curtis. 1974. Colleter anatomy in red the field, suggest that the stipules provide a physical bar- mangrove, Rhizophora mangle (Rhizophoraceae). Can. J. rier, and the colleter secretions provide a viscous fluid bar- Bot. 52: 2277-2278. rier, that protect the young shoots from the strong wind, Lersten, N.R. 1974a. Colleter morphology in Pavetta, Neorosea salt spray, tidal immersion and high temperatures that and Tricalysia (Rubiaceae) and its relationship to the bacte- SHEUE et al. — Stipules and colleters of the mangrove Rhizophoraceae 253

rial leaf nodule symbiosis. Bot. J. Linn. Soc. 69: 125-136. Sheue, C.R., H.Y. Liu, C.C. Tsai, S.M.A. Rashid, J.W.H. Yong, Lersten, N.R. 1974b. Morphology and distribution of colleters and Y.P. Yang. 2009a. On the morphology and molecular and crystals in relation to the and bacterial leaf basis of segregation of two species Ceriops zippeliana nodule symbiosis of Psychotria (Rubiaceae). Amer. J. Bot. Blume and C. decandra (Griff.) Ding Hou (Rhizophoraceae) 61: 973-981. from southeastern Asia. Blumea 54: 220-227.

Mangalan, S., K.P. Kurien, P. John, and G.M. Nair. 1990. De- Sheue, C.R., H.Y. Liu, C.C. Tsai, and Y.P. Yang. 2010. Compari- velopment, structure and cytochemistry of resin-secreting son of Ceriops pseudodecanda sp. nov. (Rhizophoraceae), a colleters of Gardenia gummifera (Rubiaceae). Ann. Bot. 66: new mangrove species in Australasia, with related species. 123-132. Bot. Stud. 51: 237-248. Parani, M., C.S. Rao, N. Mathan, C.S. Anuratha, K.K. Narayan- Sheue, C.R., H.Y. Liu, and Y.P. Yang. 2003a. Morphology on an, and A. Parida. 1997. Molecular phylogeny of man- stipules and leaves of the mangrove genus Kandelia (Rhizo- groves: III. Parentage analysis of a Rhizophora hybrid using phoraceae). Taiwania 48: 248-253. random amplified polymorphic DNA and restriction frag- Sheue, C.R., H.Y. Liu, and J.W. H. Yong. 2003b. Kandelia obo- ment length polymorphism markers. Aquat. Bot. 58: 165- vata (Rhizophoraceae), a new mangrove species from East- 172. ern Asia. Taxon 52: 287-294. Roshchina, V.V. and V.D. Roshchina. 1993. The Excretory Func- Sheue, C.R., Y.P. Yang, H.Y. Liu, F.S. Chou, S.C. Chang, P. tion of Higher Plants. Springer-Verlag, Berlin, Germany. Saenger, C.P. Mangion, G. Wightman, J.W.H. Yong, and Schwarzbach, A.E. and R.E. Ricklefs. 2000. Systematic affinities C.C. Tsai. 2009b. Reevaluating the taxonomic status of Ce- of Rhizophoraceae and Anisophylleaceae, and intergeneric riops australis (Rhizophoraceae) based on morphological relationships within Rhizophoraceae, based on chloroplast and molecular evidence. Bot. Stud. 50: 89-100. DNA, nuclear ribosomal DNA, and morphology. Amer. J. Sheue, C.R., J.W.H. Yong, and Y.P. Yang. 2005. The Bruguiera Bot. 87: 547-564. (Rhizophoraceae) species in the mangroves of Singapore, Setoguchi, H., K. Kosug, and H. Tobe. 1999. Molecular phylog- especially on the new record and the rediscovery. Taiwania eny of Rhizophoraceae based on rbcL gene sequences. J. 50: 251-260. Plant Res. 112: 443-455. Thiebaut, L. and P. Hoffmann. 2005. Occurrence of colleters in Shah, J.J. and K.P. Sundarraj. 1965. Stipular sclereids in Rhizo- Erythroxylaceae. Kew Bull. 60: 455-459. phora mucronata L. Curr. Sci. 5: 155-156. Thomas, V. and Y. Dave. 1991. Comparative and phylogenetic Sheue, C.R. 2003. The comparative Morphology and Anatomy significance of the colleters in the family Apocynaceae. of the Eastern Mangrove Rhizophoraceae. Ph. D. Disserta- Feddes Repert. 102: 177-182. tion. National Sun Yat-sen University, Kaohsiung, Taiwan. Tomlinson, P.B. 1986. The Botony of Mangroves. Cambridge Univ. Press, Cambridge. 254 Botanical Studies, Vol. 53, 2012

紅樹科紅樹林植物的托葉與指狀腺體的形態與構造之比較研究

許秋容1 陳映如1 楊遠波2,3

1 國立中興大學 生命科學系 2 國立中山大學 生物科學系 3 大葉大學 生物資源學系

紅樹科紅樹林植物的枝稍具有圓形或扁平之托葉，緊密包圍著幼葉與頂芽。此托葉向軸面基部的指 狀腺體分泌出明顯可見之樹脂狀至乳狀物質浸潤幼芽。本文首次系統性地觀察及比較本科紅樹林植物的 18 個分類群之托葉與指狀腺體的形態與構造。托葉內三種形態的厚壁異形細胞 ( 細蕊紅樹與紅樹屬 )、 發達的厚角組織或與表皮細胞外之厚角質層，提供了機械性支持的結構基礎。數十個至數百個 (35-580) 似手指狀之指狀腺體聚集呈特定的形狀，具屬的專一性：紅茄苳屬呈長方形至梯形、細蕊紅樹屬和水筆 仔屬為三角形 、紅樹屬則呈帶狀。各托葉內指狀腺體的排數、總數與指狀腺體的個別大小，在各分類 群並不相同，具分類價值。依據托葉和指狀腺體的特徵顯示細蕊紅樹屬和水筆仔屬的關係最接近，而紅 樹屬則可能呈現較後起的特徵。本群植物的指狀腺體儘管在形態上呈現短柄桿狀 ( 紅茄苳屬 )，長柄桿 狀 ( 細蕊紅樹屬和水筆仔屬 ) 或無柄漸尖桿狀 ( 紅樹屬 )，但其解剖構造均為標準型。此腺體的中心軸由 細長的薄壁細胞構成，其外圍似柵狀的薄壁表皮細胞具分泌功能，與中心薄壁細胞呈垂直排列。由托葉 所具的結構與機械性的保護功能，和其內指狀腺體的生理浸潤作用，可能對此紅樹林植物的頂芽提供了 重要的保護，以助其應付嚴酷的環境。

關鍵詞：解剖；紅茄苳屬；細蕊紅樹屬；指狀腺體；水筆仔屬；紅樹林；紅樹屬；標準型；厚壁細胞； 托葉。